The present invention is directed to methods for the tissue culture and transformation of elite inbreds of corn (Zea mays L.). More specifically, the present invention is directed to a method for initiating Type II callus from corn tissue. The present invention is also directed to a method for enhancing the integration of foreign DNA in the transformation of corn using a heat shock treatment. The present invention is further directed to a method of transforming elite inbreds of corn using Agrobacterium.
The publications and other materials used herein to illuminate the background of the invention, and in particular, cases to provide additional details respecting the practice, are incorporated by reference, and for convenience are referenced in the following text by author and date and are listed alphabetically by author in the appended bibliography.
Cells which undergo rapid division and are totipotent are generally regarded as highly suitable targets for introduction of DNA as a first step in the generation of transgenic plants. In corn, one prolific source of such cells is the so-called Type II callus (Armstrong and Green, 1985). Initiation of this type of callus has been achieved using immature embryos of certain non-elite corn inbred lines, most notably A188. Hybrid embryos with this inbred as a parent have also been used successfully. There are, however, no reports of high frequency initiation of Type II callus from immature embryos or other tissue of elite corn inbreds. Therefore, it is desired to develop a method for culturing tissue of elite corn germplasm which results in reproducible and high frequency initiation of Type II callus from elite corn germplasm.
Introduction of genes into corn can be accomplished in several ways e.g. (a) particle bombardment of cultured cells (Gordon-Kamm et al., 1990), immature embryos (Koziel et al., 1993), meristems (Lowe et al., 1995), (b) electroporation of immature embryos (D""Halluin et al., 1992), cultured cells (Laursen et al., 1994), (c) electroporation and/or polyethylene glycol treatment of protoplasts (Rhodes et al., 1988; Omirulleh et al., 1993), and (d) co-cultivation with Agrobacterium tumefaciens (Ishida et al., 1996; Hei and Komari, 1997; Zhao et al., 1998). Agrobacterium tumefaciens-mediated DNA delivery has a number of advantages. Firstly, the time taken to produce transgenic plants is short when compared to other methods. Secondly, transgenes are generally inserted as single copies, increasing the efficiency with which usable breeding material can be produced. Thirdly, high efficiencies of DNA delivery can be achieved. For breeding purposes it would be ideal to introduce genes via Agrobacterium tumefaciens directly into elite corn lines.
Following introduction of foreign DNA into target cells and subsequent cell division, selection is applied to identify those cells in which integration and expression of the foreign DNA is occurring. Any procedure which could increase the frequency with which foreign DNA integrates and expresses would greatly improve transformation protocols. A procedure for increasing the efficiency of integration of DNA into elite corn gerniplasm is described herein.
Initial methods of Agrobacterium-mediated corn transformation which were developed, while effective for some germplasm, do not allow for efficient transformation of elite lines. Hei et al. (European Published Patent Application No. 604 662 A1) discloses a method for transforming monocotyledons using Agrobacterium. In this method, plant tissues were obtained from the monocotyledon maize and the tissues were exposed to Agrobacterium during the tissue differentiation process. Hei et al. disclose a maize transformation protocol using maize calli. Saito et al. (European Published Patent Application No. 672 752 A1) disclose a method for transforming monocotyledons using the scutellum of immature embryos. Ishida et al. (1996) also disclose a method specific for transforming maize by exposing immature embryos to A. tumefaciens. The methods were optimized for inbred A188 maize lines. Transformation frequencies ranged from 12% to 30% at their highest for immature embryos from A188 lines that were 1.0-1.2 mm in length. Maize lines derived from crosses of A188 had significantly lower transformation frequencies ranging from 0.4% to about 5.3%. A188 is not generally considered a commercially useful line and Ishida et al. (1996) failed to obtain recovery of stable transformants in lines other than those containing A188.
In a subsequent method of Agrobacterium-mediated corn transformation (Zhao et al., 1998), efficient transformation of elite lines was possible using non-LS salt medium for the tissue culture steps, including the steps of contacting and co-cultivating immature embryos with Agrobacterium. This method achieves high transformation frequency of hybrids between elite lines and A 188 (e.g., a A188xc3x97B73 hybrid), a result which, although higher, is similar to the transformation frequency achieved in the initial transformation procedures. Although the transformation frequency of Pioneer elite inbreds (0.6-14.4%, with an average transformation frequency of 2.6%) was lower than that achieved for the hybrids, this method did result in the transformation of elite corn inbreds.
Thus, it is desired to develop methods which allow for the more efficient transformation of elite lines, i.e., methods which allow for the introduction of genes into elite corn lines at very high efficiency using Agrobacterium tumefaciens-mediated DNA delivery.
The present invention is directed to methods for the tissue culture and transformation of elite inbreds of corn (Zea mays L.). More specifically, the present invention is directed to a method for initiating Type II callus from corn tissue. The present invention is also directed to a method for enhancing the integration of foreign DNA in the transformation of corn using a heat shock treatment. The present invention is further directed to a method of transforming elite inbreds of corn using Agrobacterium.
In accordance with one embodiment of the present invention, Type II callus is initiated from corn tissue, preferably immature embryo, by adding a monosaccharide to the callus initiation medium. The preferred monosaccharide is glucose.
In accordance with a second embodiment of the present invention, the integration of foreign DNA into corn tissue in transformation of corn tissue is enhanced by application of a heat shock treatment following contact of the foreign DNA with the corn tissue. In a preferred embodiment, the heat shock is conducted at a temperature of 45xc2x0 C. for about 20 to about 90 minutes, preferably about 30 to about 60 minutes, more preferably about 30 minutes, at a time from about 24 to about 72 hours after the contact of the DNA with the corn tissue, preferably from 48 to 54 hours.
In a third embodiment of the present invention, the transformation of elite corn inbreds is achieved at a high frequency by co-cultivating corn tissue with Agrobacterium tumefaciens. In one particular embodiment, the transformation of elite corn inbreds is enhanced by using Agrobacterium freshly grown from glycerol cultures stored at about xe2x88x9286xc2x0 C. In a second specific embodiment, the frequency of transformation is enhanced by co-cultivating corn tissue and Agrobacterium at 19xc2x0 C. In a third particular embodiment, the frequency of transformation is enhanced by using lower levels of cefotaxime in the culturing media. In a further embodiment, the frequency of transformation of elite corn inbreds is enhanced using a combination of all of these techniques.